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Release v3.3.2: November 2015© Copyright by Amplyus LLC, all rights reserved
DNA amplification and analysis:miniPCRTM Crime Lab
Science for everyone, everywhere
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Welcome!
Our goals for today
1. Review DNA structure and DNA amplification concepts
2. Solve a crime mystery using PCR!
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Bringing DNA science to more places
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PCR is at the heart of DNA analysis
PCR
Text
Text
Forensics
Food and agriculture
Consumer genomics
Molecular diagnostics
Personalized medicine
Human evolution
Text Text
Text
Text
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Sourc
e:
NYTim
es.
com
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Today, use DNA analysis to solve a Crime Mystery
miniPCR Crime Lab
Real-world scenario
Human genetics
Aligns with standards
Essential biotech skills
PCR DNA electrophoresis Visualization
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MicropipettingDNA
visualizationPCR
Gel electrophore
sis
Students use essential molecular techniques in a
crime mystery
45 min – miniPCR™
miniPCR Crime Lab in a 90-min class block
45 min – blueGel™
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Polymerase Chain Reaction (PCR)
A process that identifies and copies (amplifies) a specific sequence of DNA
Complex DNA sample
Amplified DNA (Billions of
copies)
Sequence ofinterest
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PCR relies on DNA’s unique structure
Source: US National Library of Medicine, NIH, Thinkquest
DNA: a double helix...DNA: a double helix......held together by base
complementarity...held together by base
complementarity
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How PCR works: 3 steps to copy DNA
Denaturation
1 94°C
Annealing
250-60°C
Primer 2
Primer 1
Extension
372°C
Taq DNA polymerase dNTPs
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How PCR works: repeat the cycle
Denaturation
Annealing Extension
DNA + primers
denatured DNA
DNA + copy
94° C
50-60° C
72° C
Repeat x ~25-30 cycles
Single molecule ~1B copies
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Missy Baker Gone Missing!
FreeBoston metroMissy Baker, pastry shop owner goes missing,
police at a lossThe mysterious disappearance of the baker raises serious concerns within the population. An enigma that befuddles police investigators.
With two abduction suspects in custody, local students volunteer to try to find the missing baker.
Boston, October 20th 2015. Husband Ned reported Missy "Sugar-Cup" Baker missing, fretful after not finding her at the shop following his daily nap. The couple resides in the apartment above the pastry shop at 2 Middleborough Rd. a popular fixture in this usually quiet neighborhood. Anxiety takes hold in the community. Quickly following the report of the missing baker (wheat-blond and thin as a stick) investigators identified two suspects, but both have remained silent after harsh interrogation.
Wooly mammoth p.5
finally clonedTransgenic kiwi p.7
solves world hunger
Following extensive police searches, investigators found blond hair strands in each of their cars.
A godsend for science students, who volunteer with high-tech DNA analysis equipment to identify the kidnapper.
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CFTR: Cystic Fibrosis Transmembrane Regulator
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CFTR mutations affect water balance in lung airway lining
Healthy CFTR protein
Inside of lungepithelial cell
Outside ofepithelial lining(lung airway)
Chlorideions
H2O molecules enter lung mucus by osmosis, hydrating it
CFTR absent or defective,affecting Chloride transport
Lung airwaywith Cystic Fibrosis
Lung airway of Healthy person
Mucus becomes too sticky, difficult to clear,
and traps bacteria
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Missy Baker has a deletion mutation in her CFTR gene
CFTR Gene
CFTR1 primer
CFTR2 primer
Healthy person: the amplified DNA fragment always has the same size, corresponding to the distance between the PCR primers
Healthy gene(most people)
CFTR1 primer
CFTR2 primer
Person carrying a CFTR deletion: the amplified DNA fragment from the mutated gene is smaller than in most (healthy) individuals
Deletion mutation(very rare)
Short PCR fragment CFTR Gene mutated CFTR Gene Deletion
Missy Baker
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Experimental set up
A. Hair DNA sample from suspect A’s car
Tube A Tube BFORENSIC SAMPLES
B. Hair DNA samplefrom suspect B’s car
Tube H Tube D
D. Control DNA from CFTR deletion mutant
H. Control DNA from healthy CFTR gene
Reference lab controls
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1. Template DNA to be amplified
2. Pair of DNA primers
3. DNA polymerase
4. dNTPs
5. Buffer to maintain pH and provide Mg2+
What goes in a PCR experiment
CFTR1 primer
CFTR2 primer
Taq
AA
A
A AA
A
TT
T
T
G
T
T
C
C
C
C
C
C
C G
GG
GG
G G
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Prepare 4 PCR tubes (200µL tubes)
DNA from hair found in Suspect A’s car
15 µL
10 µL
5 µL
A
DNA from hair found in Suspect B’s car
B
Control DNA from
Healthy CFTR gene
H
Control DNA from CFTR Deletion
mutant
D
EZ PCR Master Mix
Primer mix
Also add your initials to side of tube
Template DNA
1
2
3
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Programming PCR parameters
Initial denaturation: 94°C 30 seconds
Denaturation: 94°C 5 secondsAnnealing 57°C 5 secondsExtension 72°C 8 seconds
x30 cycles
Final extension 72°C 30 seconds
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Programming
1
2
3
30 5 5 8 30
57.0
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How miniPCRTM enables DNA amplification
1
3
4
1. Heated lid• Prevents
condensation22. Heating block
• Separates DNA strands , preparing them for copy
3. Cooling fans• Cools DNA, priming it
for copy
4. Microprocessor• Stores and controls
temperature cycles
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Monitoring DNA amplification through miniPCR™ software
What is happening to DNA molecules at each PCR step?• Denaturation• Annealing• Extension
Why do we need to add an enzyme (Taq polymerase)?• What temperature is optimal for most enzymes? • What makes Taq unique?
How many more molecules of DNA will we have with each PCR cycle?• And at the end of the entire PCR reaction?• We call this exponential amplification
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Quiz: Which of these are NOT characteristics of PCR primers?
A. Short synthetic oligonucleotide
B. Typically 18-25 bases in length
C. Double stranded DNA
D. Unique homology to the DNA template
E. Sequence with ~50% G:C content
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Next step: gel electrophoresis to visualize PCR products1. Pour an agarose gel 2. Load the PCR products
4. Visualization in a transilluminator
3. Electrophoresis
e-
- Pole
+ Pole
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21st century DNA gel electrophoresis
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Questions to probe deeper – I (During gel run)
What is the subcellular location of the CFTR gene product?
How many different types of CFTR mutations can cause cystic fibrosis?
How common do you think CFTR mutations are?
Are these mutations typically dominant? Recessive?
What would we see if Missy Baker were heterozygous for the CFTR deletion?
What can be done to treat people with CFTR gene mutations?
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How did the investigation turn out?
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Questions to probe deeper – II (After gel run)
How do you think the investigation turned out?
Is the evidence consistent with suspect A being guilty? Why?
Is the evidence consistent with suspect B being innocent? Why?
What’s the importance of running controls from the Reference Lab?
What caveats should be applied when analyzing forensic DNA evidence?
What was the most unexpected thing you learned today?
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miniPCR in real-life forensics: Amplification of STRs used in human identification
Humansamples
Fluorescence detection
PCR (miniPCR or
ABI 9700
Capillary electrophore
sis
Short tandem repeat (STR) data obtained by Signature Science LLC and used with permission. Mini8 = miniPCR; 9700 = ABI 9700 thermal cycler
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SamplesVisualizatio
nPCR
Electrophoresis
Hope you’ve enjoyed the lab!
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We hope you enjoyed the lab! Questions?
www.miniPCR.com
@miniPCR
Facebook.com/miniPCR
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Additional resources
Interpreting CFTR mutationshttp://ghr.nlm.nih.gov/gene/CFTRhttp://www.cftrscience.com/
The Innocence Projecthttp://www.innocenceproject.org/
History of the use of DNA in crime solvinghttp://www.forensicmag.com/articles/2005/01/evolution-dna-evidence-crime-solving-judicial-and-legislative-history
Deutsche Welle: 30 years of DNA Fingerprintinghttp://www.dw.de/from-paternity-to-criminal-cases-dna-fingerprinting-has-been-30-years-of-eureka/a-17911987
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What are the effects of Cystic Fibrosis (CF) genetic mutations?
Cystic Fibrosis can be caused by one or more mutations in the CFTR gene, which encodes a channel protein involved in the passage of chloride ions through the cell membrane.
The defective gene interferes with the body’s ability to transfer water and salt to and from cells. This causes secretions, which are normally thin and watery in healthy people, to become very thick and sticky.
The thick secretions clog up organs and prevent them from working properly. An increase in the viscosity of cell secretions often causes respiratory diseases, which are the main cause of death from CF.
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Going deeper: Medically relevant CFTR mutations
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Appendix: PCR cycles amplify DNA exponentially
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Alternative approach: 2 PCR Tubes per groupTwo PCR tubes per group
A. Hair DNA sample from suspect A’s car
Tube “A” Tube “B”
“DETECTIVES”
Groups 1 through 4
B. Hair DNA samplefrom suspect B’s car
“REFERENCE LAB”
Groups 5 through 8
Tube “H” Tube “D”
D. Control DNA from CFTR deletion mutant
H. Control DNA from healthy CFTR gene
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Complete biotech toolkit under $1,000
$60